Xylanase degrades the polysaccharide, xylan, which is the major constitute of hemicelluloses in plants. Xylan is most abundant renewable resource next to cellulose in the world, which is a hetero-polysaccharide having β-1,4-D-pyranoxylose-linked backbone and various substituted side chains. Due to its complicated structure, it needs an enzyme-degrading system for complete breakdown of xylan. These enzymes include the backbone degrading enzymes: Endo, β-1,4 xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37); and side chain degrading enzymes: α-L-arabinofuranosidase (EC 3.2.1.55), α-glucuronidase (EC 3.2.1.139), and acetylxylan esterase (EC 3.1.1.72) (Q. K. Beg, M. Kapoor, L. Mahajan, and G. S. Hoondal. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol (2001) 56:326-338) Among these enzymes, endo β-1,4 xylanase contributes for the most part of xylan degradation.
Endo-xylanases are enzymes that randomly cleave the β(1-4) linkages between xylose residues making up the backbone of xylans, a prevalent form of hemicellulose found predominantly in plant primary and secondary cell walls. Many prior arts, such as U.S. Pat. No. 5,948,667 (published on Sep. 7, 1999), U.S. Pat. No. 6,300,114 (patented on Oct. 9, 2001), U.S. Pat. No. 5,824,533 (patented on Oct. 20, 1998) and WO 93/25693 (published on Dec. 23, 1993), etc, have disclosed various xylanases and their uses. The known applications of xylanases are numerous. For instance, the treatment of forages with xylanases (along with cellulases) to increase the rate of acid production, thereby ensuring better quality silage and improvement in the subsequent rate of plant cell wall digestion by ruminants has been described. Xylanases can be used to treat rye, and other cereals with a high arabinoxylan content to improve the digestibility of cereal by poultry and swine. Xylanases can be used in bioconversion involving the hydrolysis of xylan to xylooligosaccharides and xylose which may serve as growth substrates for microorganisms. This could involve simultaneous saccharification and fermentation. Xylanases can be used in biopulping to treat cellulose pulps to remove xylan impurities or to produce pulps with different characteristics. In some cases they can be applied to reduce the amount of chlorine needed to bleach the pulp and reduce the energy needed for refining pulp. Further, xylanases are useful in the retting of flax fibers, the clarification of fruit juices, the preparation of dextrans for use as food thickeners and the production of fluids and juices from plant materials.
Commercially available xylanases and their activities and purposes are reviewed in “Q. K. Beg, M. Kapoor, L. Mahajan, and G. S. Hoondal. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol (2001) 56:326-338”.
Particularly, it was reported that the pretreatment of unbleached kraft pulp with xylanase results in a reduced consumption of chemicals for bleaching process. Prior arts have also disclosed that the xylanase pretreatment is useful in conjunction with bleaching sequences consisting of Cl2, ClO2, H2O2 and O3. As a direct result of the better bleachability of the pulp after such a xylanase treatment, there is a reduction of the subsequent consumption of bleaching chemicals, which when chloride containing chemicals are used, leads to a reduced formation of environmentally undesired organo-chlorine compounds. Also as a direct result of the better bleachability of pulp after a xylanase treatment, it is possible to produce a product with a final brightness where such brightness would otherwise be hard to achieve (such as totally chlorine free (TCF) bleaching using peroxide). Because of the substrate specificity of the xylanase enzyme, cellulose fibers are not harmed and the strength properties of the product are well within acceptable limits.
However, it is not as simple as merely adding a xylanase treatment step. Most commercial xylanases designed for pulp bleaching are not very thermotolerant, especially when neutral or alkaline pH conditions are used. In practice, xylanases are generally inefficient or inactive at temperatures higher than 60° C. Therefore, the recombinant xylanase specifically disclosed in WO 9325693, which is derived from Neocallimastix patriciarum and designated XYLA and has a specific activity of 5980 U/mg, could not satisfy the requirements of pulp and paper manufacturers.
A xylanase that is active at an alkaline pH would decrease the need to acidify the pulp prior to xylanase treatment. In addition, the temperatures of many modern kraft cooking and bleaching processes are relatively high, well above 50° C., that is unsuitable for many of the commercial bleaching enzymes. Accordingly, a need exists for thermostable xylanase preparations that are stable at alkaline pH's for use in wood pulp bleaching processes. In order to obtain thermostable xylanases, U.S. Pat. No. 6,300,114 produced proteins originating from actinomycetes in filamentous fungi such as Aspergillus or Trichoderma.
The ruminants are glorified by their ability to digest fibrous plant materials. Ruminants themselves do not produce fiber-degrading enzymes, but they harbor bacteria, fungi, and protozoa which can digest fiber to support hosts' survival (Russell, J. B., and J. L. Rychlik. 2001. Factors that alter rumen microbial ecology. Science 292:1119-22). The rumen ecosystem comprises a diverse population of anaerobic bacteria, fungi, and protozoa defined by the intense selective pressures of the ruminal environment. The ruminal microbes generally become the high activity fiber-degradation resource. Up to now, there are many fiber-degradation genes isolated from rumen (Selinger, B. L., C. W. Forsberg, and K. J. Cheng. The rumen: A unique source of enzymes for enhancing livestock production. Anaerobe 2:263-284 (1996)).
However, up to now, the xylanase relevant genes isolated from rumen were obtained by first constructing a cDNA gene data base and then screening the genes contained therein with xylan relevant bases (Durand, R., C. Rascle, and M. Fevre. Molecular characterization of xyn3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis. Curr Genet Vol. 30 Issue 6 (1996) pp 531-540). Through such a known method, the xylanase gene sequences isolated from ruminal fungi lack intron. Anyway, such a known method is quite time-consuming and inefficiency. Without constructing said cDNA gene data base, the present invention directly uses the DNA from ruminal fungi as a template and adopts a suitable specific primer to proceed with PCR. In such a way, the xylanase gene sequences can be rapidly obtained. Furthermore, some new xylanases expressed by the gene sequences obtained in this way are quite active under high temperature and alkaline reaction condition and have high specific activity. These new recombinant xylanases may be produced by prokaryotic or eukaryotic expression systems.
All references cited herein are incorporated herein by reference.